Method of dephosphorylating an endotoxin in vivo with alkaline phosphatase

ABSTRACT

The invention relates to pharmaceutical compositions suitable for treating or curing clinical complications mediated by endotoxin, including sepsis. The compositions contain components suitable for detoxifying endotoxin rendering it less deleterious to mammals such as humans, in particular to patients with reduced host-defence resistance. The invention also relates to pharmaceutical compositions suitable for stimulating bone formation, e.g. for mending broken bone or for prophylaxis or therapy of metabolic bone diseases such as osteoporosis and osteomalacia and pharmaceutical compositions for decreasing or inhibiting undesired bone formation. The pharmaceutical compositions according to the invention are directed at modulating phosphatase activity in vivo.

This application claims benefit of international PCT/NL94/00189, filedAug. 10, 1994.

FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions suitable fortreating or curing clinical complications induced by infections withGram-negative bacteria, including sepsis. In particular the invention isdirected at systemically applicable compositions. The compositionscontain components suitable for detoxifying bacterial-wall derivedlipopolysaccharides (also known as endotoxins) rendering these productsless deleterious to mammals such as humans, in particular to patientswith sepsis, optionally in combination with reduced host-defenceresistance, i.e. after organ transplantations, during leucopenia(ref. 1) associated with cancer or chemotherapeutic treatment of canceror during AIDS and AIDS-related diseases (ref. 2).

The invention also relates to pharmaceutical compositions suitable forstimulating bone formation, e.g. for mending broken bone or forprophylaxis or therapy of metabolic bone diseases such as osteoporosisand osteomalacia and also pharmaceutical compositions for decreasing orinhibiting undesired bone formation.

BACKGROUND INFORMATION

Endotoxin is a negatively charged lipopolysaccharide present in thecapsule of Gram-negative bacteria (ref. 3). Endotoxins are complexes ofphospholipid (lipid A) and polysaccharide. The endotoxins produced bydifferent bacteria differ in their antigenicity but they all have thesame biological effects which are mainly due to lipid A. For thepurposes of this description the term endotoxin also comprisesenterotoxins. In addition to the negatively charged sugar moieties, anendotoxin contains two phosphate groups which are essential for itstoxicity (ref. 3, 4).

Although it is an ubiquitous molecule in the external environment aswell as in the gastro-intestinal tract of many species, an endotoxin canbe extremely deleterious to these species once it leaves thegastro-intestinal tract e.g causing sepsis and inflammation such as inan abcess even in amounts as low as 10 picogrammes. Yet so far, noimportant endotoxin detoxifying mechanism has been found in vivo (ref.5).

Endotoxin is known to induce serious even lethal complications (ref. 5and 6). In fact, despite the use of antibiotics, this bacterial productis the major cause of death in intensive-care units in Western society.

There are numerous different endotoxins produced by variousmicroorganisms and consequently the actions of endotoxin in vivo arenumerous as are the ways it can enter the organism. The symptomsassociated with Gram negative infections therefore also vary widelyamong patients (ref. 7). These symptoms may be further complicated byseptic shock of which hypotension, peripheral vasodilation and diffuseintravascular coagulation are the main characteristics (ref. 8).Subsequently organs such as heart (acute heart failure), lungs (adultrespiratory distress syndrome). kidney (acute tubular necrosis) andbrain may be affected (ref. 8). Endotoxin mediated pathology alsocomprises the syndrome of multiple organ failure and any other syndromegenerally accepted in the art to be directly or indirectly caused byendotoxin.

To date, antibodies directed against endotoxin are the only endotoxindetoxifying proteins known to reduce toxicity irreversibly, but theclinical value of these antibodies remains to be established. Othersubstances which are able to bind endotoxin, such as lipopolysaccharidebinding protein and high density lipoproteins (HDL) (ref. 9), exhibitthe major drawback of forming reversible complexes in vivo. Upondissociation of these complexes, the native (toxic) molecule is producedagain. Furthermore although the detoxifying activity of plasma has beennoted for some time (ref. 10) efforts to isolate or characterise thesubstance(s) responsible for this activity have not been successful.Other experimental approaches to treat sepsis include the application ofpreparations which antagonize the activities of cytokines (e.g. TNF-α),which are important mediators of endotoxin-induced shock, aggravatingthe effects of endotoxin in vivo. A major disadvantage of this approachis that these preparations do not detoxify the causative agent butrather block one of the reactions of the body to this toxin. Inaddition, antagonizing naturally occurring cytokines may cause multipleside effects.

Alkaline phosphatase (EC 3.1.3.1) is a common enzyme present in manyspecies, including man and has been studied extensively. The DNAsequence encoding alkaline phosphatase has even been obtained, but sofar no commercial exploitation thereof has occurred. Although the enzymeis routinely applied as antibody label or as a marker for liver andneutrophil function, it's biological relevance is still unknown.Recombinant alkaline phosphatase enzymes with improved specific activityused as indicator reagents are disclosed, e.g. in EP-A-0 441 252. Thispatent application however mentions nothing regarding anti-endotoxinactivity or bone formation of alkaline phosphatase. The cited Europeanpatent application describes a number of derivatives in which one aminoacid differs from the wild type. The substituents include replacement ofThr 100 by Val or Ile, replacement of Lys 328 by Arg, replacement of Val99 by Ala, replacement of Thr 107 by Val, replacement of Asp 101 by Ser,replacement of Val 377 by Ala and replacement of Ser 115 by Gly as wellas replacement of Ala 103 by Asp. Other derivatives described in thecited patent application are derivatives with Mmaleimidobenzoyl-N-hydroxysuccinimide ester for carrying out a sandwichEIA and a thiolated mutant of alkaline phosphatase which can be derivedthrough use of succinimidyl-4-N-maleimidomethyl-1-thicapramidecyclohexanecarboxylate. Of all these derivatives no mention is made ofthe charge carried by the alkaline phosphatase derivative. It is pointedout that all the mentioned derivatives with the exception of thereplacement of Ala 103 by Asp have been calculated by us as resulting ina more positive netto charge or an equal netto charge in comparison tothe corresponding native alkaline phosphatase.

Alkaline phosphatase is a membrane-bound ecto-enzyme which is known todephosphorylate extracellular molecules. The enzyme is present in manyorgans, including intestine, kidney, osteoblasts and neutrophils (ref.11, 12 and 13). in vitro, it exhibits a pH optimum of approximately10.5. (ref. 12). This extremely high pH optimum has hampered recognitionof its biological relevance (ref. 12-14), because it was felt that thispH level does not occur in biological tissues of the intact organism.

In a number of publications a derivative of alkaline phosphatase andcollagen, in particular fibrillar collagen is described. Nothing ismentioned about the netto negative charge of such a derivative, however,we have calculated that the netto charge is positive in comparison to anon-derivatized alkaline phosphatase.

In U.S. Pat. No. 4,409,332 (1983) collagen sutures derivatized withalkaline phosphatase are described as reducing the inflammatorycharacteristics of collagen. The collagen induced inflammation is not aninflammatory reaction due to endotoxins, it is an inflammation that isgenerally caused by damage of tissue that has occurred, by the fact thatcollagen is a heterologous protein which is foreign to the body and bythe fact that collagen always induces coagulation in vivo which cansubsequently activate inflammatory cells in a number of manners. Aninflammation due to infections of the wound is not likely as the authorsthemselves frequently state that they worked in a sterile environment,using sterile solutions. A person skilled in the art cannot derive fromthis cited patent publication how alkaline phosphatase coupled tocollagen can inhibit the inflammation usually caused by collagen. Anumber of manners can however be postulated such as, for example byprotection of antigens for cells of the specific immunoreaction, therebyprohibiting recognition or by binding positively charged mediators ofthe non-specific immune reaction as alkaline phosphatase containsnegatively charged sugar groups. Another possibility is inhibition ofthe coagulation cascade by masking collagen or de-phosphorilation ofmediators, such as ATP, ADP and platelet activating factor or by bindingof positively charged mediators and cofactors.

In the cited document it is stated that even though the hydrolysisfunctions of outline phosphatase have intensively been studied for morethan 50 years no clear image has arisen concerning the value of theenzyme to the organism. In summary the in vivo activity of alkalinephosphatase is not clear. No link is made in the cited document betweenalkaline phosphatase and bone formation or anti-endotoxin activity. Notheoretical background is given to the anti-inflammatory activity of thecoupling of alkaline phosphatase to collagen. It is in fact questionablewhether a person skilled in the art would even attribute theanti-inflammatory activity to the presence of alkaline phosphatase orwhether the fact that specific groups of collagen are protected by thepresence of a random derivative provides the anti-inflammatory action.This can be derived from the fact that it is described that the use ofcross-linking agent, such as glutaraldehyde or other cross-linking meansappears to increase the anti-inflammatory characteristics of thematerial.

In U.S. Pat. No. 4,394,370 an anti-inflammatory complex of collagen andBMP is described as well as the use thereof in the healing of brokenbones, which is further elucidated in U.S. Pat. No. 4,409,332. U.S. Pat.No. 4,394,370 describes the use of reconstituted collagen anddimineralized bone particles or reconstituted collagen and a solubilizedbone morphogenetic protein, fabricated in a sponge for in vivoimplantation in osseous defects. Both demineralized bone particles andbone morphogenetic protein have demonstrated the ability to induce theformation of osseous tissue in animal and human experiments.Reconstituted collagen conjugate is highly biocompatible and can befabricated in a variety of configurations. This material can be used asa, grafting implant in plastic and reconstructive surgery, periodontalbone grafting and in endodontic procedures. The structural durability isenhanced by cross-linking with glutaraldehyde, which is also used tosterilize and disinfect the collagen conjugate prior to implantation. Itis stated that the soluble factor from demineralized bone, bonemorphogenetic protein is osteo inductive and it is also known that thedemineralized bone is also condusive to osteogenesis. The use ofalkaline phosphatase coupled to BMP and collagen as disclosed in U.S.Pat. No. 4,394,370 is not directed specifically at a bone formationincreasing activity of alkaline phosphatase as such but more the factthat alkaline phosphatase linked to collagen has a less inflammatorycharacter than non-derivatized collagen. U.S. Pat. No. 4,394,370 is inparticular directed at collagen BMP conjugate sponges and the use ofalkaline phosphatase conjugated to such a sponge is merely one of anumber of embodiments of BPM-collagen uses and as such is not directedat the same invention as the subject patent application namely the invivo activity of phosphatase as such or derivatives thereof amongstothers for increased bone formation. In U.S. Pat. No. 4,394,370 nomention is made of the anti-endotoxin activity of alkaline phosphatase.

WO 93/00935 describes that the possible role of the enzyme alkalinephosphatase in promoting the calcification of bone has been postulatedfor many years. However that the relevance of such in vitromineralization studies to the situation in vivo has been questioned,particularly in view of the relatively high concentrations of phosphateesters used in the in vitro studies and also because the rate ofhydrolysis of the phosphate esters and physiological pH levels would beexpected to be too low to be relevant to the process of mineralization.In the cited patent application Beertsen et al. describe thatcombination of a biocompatible carrier material, preferably one whichcan mineralize to some degree itself, such as fibrillar collagen with aquantity of a phosphatase enzyme will promote mineralization. Preferablythe combination of alkaline phosphatase with the carrier is broughtabout by incubating the carrier with the enzyme in the presence of acoupling agent capable of covalently bonding with the carrier and withthe enzyme. Suitable coupling agents are described as biotinavidin,glutaraldehyde and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide HCl. Aparticularly preferred coupling agent is known assuccinimidyl-s-acetyl-thioacetate (SATA) in combination with maleimidohexanoyl-N-hydroxysuccinimide (MHS) wherein the carrier is incubatedwith SATA and the enzyme with the MHS. The products of these twoincubation processes are combined and allowed to react to produce animplant material. The cited document describes that the coupling ofalkaline phosphatase to collagen improves the osteogenesis when such acomplex is placed in situ of the wound. The alkaline phosphatase is usedin combination with a product already known to stimulate bone formation.No description is given of use of alkaline phosphatase as such or as aderivative with a particular altered charge. It is pointed out that aderivative of alkaline phosphatase with fibrillar collagen has anincreased positive charge in comparison to non-derivatized alkalinephosphatase. A derivative of alkaline phosphatase with fibrillarcollagen is not suitable for systemic application as fibrillar collageninduces intravascular platelet activation leading to embolisms.Therefore, a complex of fibrillar collagen and alkaline phosphatasecould not be used in a method for treating osteoporosis or osteomalaciaor any other bone defect which requires systemic application. It canonly be used when immobilized in situ at the location of a wound.

DESCRIPTION OF THE INVENTION

The subject of the present invention is based on our finding thatalkaline phosphatase as such is endowed with phosphatase activityregulating certain vital body functions, even at physiological pHlevels, i.e. in vivo without having to be derivatized.

The basis for this insight was provided by the idea that at themolecular level in vivo, an alkaline micro-environment could presentitself differently from aqueous solutions in vitro. In vivo negativelycharged molecules may act as weak bases by their ability to bind H⁺.Consequently these anions induce a local increase in the pH levelthereby providing a micro environment with a pH value sufficient for thealkaline phosphatase to function as phosphatase.

The addition of negative charges can be provided in vivo in threedifferent ways: addition of net negatively charged substrates to theenzyme, secondly providing the phosphatase with a membrane carrier withnegative charges and thirdly by changing the ionization of chargedgroups in the protein itself and/or introducing negatively chargedmoieties in the protein backbone or removing potentially positivelycharged groups in the protein.

These different mechanisms either alone or in combination may explainthe unusual high pH optimum of the enzyme in vitro.

Similar considerations could be applicable to other types ofphosphatases.

Another aspect of the present invention is based on the even morespecific finding that alkaline phosphatase as such is also endowed withendotoxin detoxifying activity, even at physiological pH levels.

Endotoxin having negatively charged moieties can thus for example supplynegatively charged residues necessary in the alkaline phosphatase'smicro environment. In this way, this ubiquitous enzyme can provideprotection in vivo against endotoxin, the ubiquitous product of Gramnegative bacteria.

The use of enzyme preparations to detoxify endotoxin itself has theadvantage that treatment of the disease in the early stage is possibleand has the further advantage of irreversibly reducing toxicity of theendotoxin. In addition as activity of enzymes is substrate specific theside-effects of using an enzyme such as alkaline phosphatase arelimited.

The phosphate groups in the lipid A moiety of endotoxin determine thetoxicity of this bacterial component. Whilst dephosphorylated lipid Amolecules retain some of their immunostimulatory activities it thusbecomes possible to make a vaccine.

A vaccine for preventing pathology mediated by endotoxins of gramnegative bacteria, said vaccine comprising phosphatase as such or aderivative of phosphatase, said derivative having phosphatase activityas active component and any adjuvant commonly used in a vaccine is anembodiment of the subject invention. Any type of derivative generallyacceptable in the field of vaccines can be used.

Yet a further aspect of the invention is directed at the use of alkalinephosphatase as such as active component in a pharmaceutical compositionfor treatment or prophylaxis requiring increasing bone formation e.g.for stimulating the mending of broken bones or in particular fortreatment and/or prophylaxis of metabolic bone diseases such asosteoporosis and osteomalacia. The negative charges necessary foroptimal alkaline phosphatase activity in vivo may not only be providedby the substrate but also by the micro-environment, or both.

In bone for instance, alkaline phosphatase is found in an extracellularenvironment rich in glycosaminoglycan (GAG)-chains, osteopontin,osteocalcin and bone sialo protein. All of these molecules areparticularly characterized by their high content of negatively chargedresidues (ref. 15). To date, the role for alkaline phosphatase in boneformation is obscure. However, its significance may be reflected by thefact that the enzyme is present at sites of bone formation (e.g. in theextracellular space between two sites of a bone fracture (ref. 16 &personal observations). Moreover, a disease like hypophosphatasemia,characterized by low levels of alkaline phosphatase activity in bone andserum is associated with skeletal deformations (ref. 17).

Again, the substrate for alkaline phosphatase is unknown, mainly becauseof the unphysiological high pH optimum of the enzyme using varioussubstrates. However, in the light of our idea it is easily conceivablethat when a given phosphorylated substrate becomes attached to stronglynegative molecules, the proper micro-environment is created. In such acondition, alkaline phosphatase may express optimal activity atphysiological pH levels, and dephosphorylate this substrate therebycontributing to the formation of insoluble calciumphosphate-complexes,an important constituent of bone matrices.

Alkaline phosphatase or a derivative thereof may locally dephosphorylateorganic phosphate esters (of yet undefined nature) and thus contributeto the formation of calcium phosphate-complexes. In addition, since highconcentrations of inorganic phosphate in the extracellular environmentare necessary to create the optimal physico-chemical conditions forbone-mineralization (a high ionic strength facilitates precipitation ofphosphate-complexes), plasma alkaline phosphatase or a derivativethereof activity may increase inorganic phosphate concentrations inblood and thus further contribute to the process of bone formation.Therefore, alkaline phosphatase or a derivative thereof apparently has adual effect in vivo: it directly causes mineralization of the bonematrix and it further facilitates this process by causingsupersaturation of the environment with phosphate.

Recent experimental data support this notion regarding the role of aderivative of alkaline phosphatase in vivo during bone formation. Sheetsof collagen layers coated with an alkaline phosphatase derivative arerapidly mineralized when implanted subcutaneously in rats (ref. 32). Thedegree of mineralization depends on the amount of alkaline phosphatasederivative bound to the collagen implants, and on the serum levels ofinorganic phosphate in these animals. Older female rats (35 weeks ofage) showed lower serum levels of Pi and a lower degree ofmineralization of the implants as compared to young male rats. Inaddition, dietary phosphate deprivation in normal mice leads to impairedbone formation (ref. 33). Also in vitro, mineralization of collagensheets coated with the alkaline phosphatase derivative could beattenuated by reducing Pi concentrations in the medium.

Metabolic bone diseases such as osteoporosis and osteomalacia generallyaffect the elderly man. Conflicting reports exist as to the questionwhether serum alkaline phosphatase activity is increased, decreased orunchanged in elderly people (for a review see ref. 34). It is known thatserum alkaline phosphatase activity may not reflect tissue-boundalkaline phosphatase activity adequately (ref. 34). In fact, severalreports suggest a gradual decrease in alkaline phosphatase activity infor instance hepatic cells (ref. 35) intestina and leucocytes (ref. 36)with age. It may be speculated that metabolic bone diseases likeosteoporosis and osteomalacia are characterized by an impaired activityof alkaline phosphatase or a derivative thereof in situ, caused eitherby a local lack of enzymes or by a reduced amount of negatively chargedmolecules within the micro-environment. The higher incidence ofosteoporosis in the female population may also be explained by thenotion that alkaline phosphatase or a derivative thereof is involved inthe pathogenesis of this disease: the activity of alkaline phosphataseor a derivative thereof is, at least in women, regulated by hormonessuch as estradiol (ref. 37), as is also demonstrated by the rise inserum alkaline phosphatase activity during pregnancy (ref. 18). In themenopause this regulating system disintegrates.

It is conceivable that a lower serum alkaline phosphatase activitycauses a lower concentration of Pi in blood and that this condition canbe reversed by administration of (alkaline) phosphatase or a derivativethereof with (alkaline) phosphatase activity with a long circulatinghalf-life. In the light of the above mentioned, bone mineralization maybe further enhanced by coupling (alkaline) phosphatase to negativelycharged molecules and/or by increasing the intrinsic anionic charge of(alkaline) phosphatase. Administration of (alkaline) phosphatase assuch, or stimulating (endogenous (alkaline)) phosphatase activity and/orproduction, may therefore be beneficial to patients with metabolic bonediseases such as osteoporosis or osteomalacia. Systemic application of(alkaline) phosphatase as such as active component of a pharmaceuticalcomposition to persons with (multiple) bone fractures and apharmaceutical composition comprising (alkaline) phosphatase as such asactive component also fall within the scope of the present invention. Inaddition, inhibition of (alkaline) phosphatase activity may be an optionfor therapy in patients with malignancies characterized by excessivebone-formation, such as osteosarcoma, or secondary tumours derived frommetastatic carcinoma's. The high alkaline phosphatase activity found inosteosarcoma tissue (ref. 19) may therefore not only be a diagnosticmarker for increased bone formation but an entrance for therapeuticintervention as well.

The subject invention is therefore also directed at a method oftreatment of pathology associated with rapid bone formation such asosteosarcoma, said method comprising decreasing or inhibiting alkalinephosphatase activity, preferably in a target specific manner i.e. at thelocation where said pathology occurs. The decrease in phosphataseactivity can be brought about e.g. by lowering formation of phosphataseor by competitively binding phosphatase preventing it'sdephosphorylation. A pharmaceutical composition comprising at least onesubstance capable of decreasing or inhibiting phosphatase activityand/or the concentration of (alkaline) phosphatase (activity), saidsubstance preferably being targeted to act at a location where undesiredbone formation is to be prevented also falls within the scope of theinvention.

A pharmaceutical composition according to the invention is preferablynon-toxic in the circulatory system and thus systemically acceptable andapplicable. This allows the use against endotoxin and metabolic bonediseases and also eliminates the need for surgery in some cases wherebroken bones need mending.

To test the hypothesis that alkaline phosphatase is a protective enzymeof the host-defence system by its ability to detoxifylipopolysaccharides, we investigated whether alkaline phosphatase isable to dephosphorylate endotoxin of Escherichia coli at physiologicalpH levels. Alkaline phosphatase activity was explored in 4%formalin-fixed cryostat sections (4 μm) of intestine, kidney and spleenaccording to standard histochemical methods at alkaline (ref. 20) andphysiological pH levels (ref. 21), using either the conventionalsubstrate β-glycerophosphate (6.0 mg/ml) or endotoxin from Escherichiacoli (0.55 mg/ml; serotype 0.55:B5, Sigma Chemical Co, St.Louis,U.S.A.). At alkaline pH level, the histochemical method of Gomori (ref.20) was applied, whereas the method of Wachstein and Meisel (ref. 21)was used at the lower pH level. Sections were incubated with substratefor one hour at 37° C. At pH 7.4 and 9.0, phosphate precipitates,indicating enzyme activity, were found in intestine and kidney sectionswhen endotoxin was used as substrate (FIG. 1). In the spleen, strongpositive cells scattered throughout the red pulpa were found.Distribution of reaction product was identical for both substrates. Allsections incubated without substrate were completely devoid of reactionproduct. In addition, the selective alkaline phosphatase inhibitorlevamisole (1.0 mM) (ref. 20) completely inhibited phosphate releasefrom endotoxin in kidney sections, whereas in intestine sections thisactivity was attenuated by the well known inhibitor of intestinalalkaline phosphatase, L-phenylalanine (5 mM) (ref. 22), but not by thestereoisomer D-phenylalanine (5 mM). Thus, both distribution of enzymeactivity in various organs, as well as results obtained with selectiveinhibitors demonstrate that endotoxin is dephosphorylated by alkalinephosphatase at physiological pH levels.

The pH optimum of alkaline phosphatase activity was studied in a morequantitative way using tubular brushborder fragments of the rat kidney.This particular enzyme preparation has the advantage that it can bestudied in association with the plasma membrane. Moreover, it can becompletely inhibited by levamisole. Preparations of tubular brushborderfragments (isolated from the cortex of PVG rat kidneys using a sieve of180 mesh and rinsed in 0.9% saline) which contained 12 μg protein(specific phosphatase activity: 86 U/mg, as assessed at pH 9.8) wereadded to 250 μl 2-amino-2-methyl-1,3-propanediol buffer of various pHlevels. The buffer contained either endotoxin from E. coli (1.25 mg/ml)or paranitrophenolphosphate (0.5 mg/ml; pNPP). 2 mM MgCl₂ was addedshortly before the start of the incubation period. After one hourincubation at 37° C., inorganic phosphate concentrations were assessedaccording to the method of Chandrarajan (ref. 23). With the conventionalsubstrate pNPP a steady increase in phosphate release along with the pHwas observed (FIG. 2, upper left corner); however, when endotoxin wasapplied as substrate, enzyme activity reached a maximum at pH 8.8 andremained stable at this level. Endotoxin- as well aspNPP-dephosphorylation was inhibited by the alkaline phosphataseinhibitor levamisole (0.2 mM). Activity at high pH levels was nothampered by de-acetylation of fatty acyl chains of endotoxin occurringat alkaline conditions (ref. 24), since one hour pre-incubation ofendotoxin at pH 9.8 did not inhibit dephosphorylation at pH 7.4 astested histochemically. Thus, in contrast to the high pH optimum foundwith the substrate pNPP, alkaline phosphatase reaches maximal activityat a less extreme pH level when endotoxin is used as substrate. It maybe speculated that the pH optimum is even lower when the enzyme isstudied in vivo within its proper micro environment comprising thenecessary additional negative charges to mimic the alkaline pH optimumobserved in vitro.

To study the effect of alkaline phosphatase upon endotoxin toxicity,Limulus assays were performed. This Litulus assay is the standard methodto assess endotoxin concentrations in vitro, based upon toxicity of themolecule towards the horseshoe crab Limulus polyphemus (ref. 25).Endotoxin (2.0 ng/ml) was incubated for one hour at 37° C. in RPMI-1640buffer (pH 7.6), together with tubular fragments (0.8 μg protein/ml,specific phosphatase activity 86 U/mg). Control samples lacked eitherendotoxin or tubular brushborder fragments. Subsequently the Limulusassay was performed. Results show a significant reduction in endotoxinconcentrations as measurable by this method in suspensions containingendotoxin and alkaline phosphatase activity, as compared to suspensionscontaining equal amounts of endotoxin without the enzyme (Table 1). Itcan be concluded that alkaline phosphatase is able to attenuate thetoxicity of endotoxin molecules at physiological pH levels, as assessedin vitro.

A further method at which the invention is a method for rendering asubstance free of toxic effects of endotoxin comprising subjecting theproduct to the phosphatase activity of phosphatase, e.g. alkalinephosphatase or a derivative of phosphatase having phosphatase activity.Such a derivative can for example be a derivative as described herein.The substance to be detoxified must be subjected to the presence of thephosphatase activity during a sufficient length of time to detoxify anyendotoxin present. A person skilled in the art will be able to ascertainwhat length of time this can suitably be without undue experimentation.

TABLE 1 Endotoxin concentrations as measured by the Limulus assay withand without pre-treatment with alkaline phosphatase [endotoxin] samplepg/ml Endotoxin 34.0 ± 13.00 Endotoxin + alkaline phosphatase <0.05 ±0    Buffer 11.3 ± 9.8 

The toxicity of endotoxin treated with alkaline phosphatase was alsostudied in vivo, taking advantage of the fact that local inflammationfollowing two successive endotoxin injections (the localShwartzman-reaction (ref. 26)) can readily be quantified. If thedetoxifying hypothesis were valid, this inflammatory reaction should bereduced after administration of endotoxin preparations pre-treated withalkaline phosphatase. Therefore, we elicited a local intradermalShwartzman-reaction and treated the second endotoxin dose with tubularbrushborder fragments at physiological pH. Influx of oxygen free radicalproducing cells, an important feature of the Shwartzman-reaction, wassubsequently examined histochemically. Thus, the Shwartzman-reaction waselicited by two successive injections of endotoxin (from E. coli 055:B5)divided by 20 hours in female PVG rats (200 g). The first endotoxininjection (1 mg/kg b.w.) was administered intravenously, whereas thesecond injection consisted of an intradermal administration of a mixtureof 70 μl RPMI 1640-medium (pH 7.6) supplemented with 2 mM MgSO₄ and 40μg endotoxin (E) or MgSO₄ alone (C). Prior to injection, media wereincubated (1 hour, 37° C.) with 6 μg tubular brushborder fragmentscontaining 86 U/mg alkaline phosphatase activity (A), with or withoutthe alkaline phosphatase inhibitor levamisole (L; final concentration1.0 mM). Control media were supplemented with saline (S) and lackedeither endotoxin, or alkaline phosphatase, or both. Two hours after theintradermal injections, dermal sites were analyzed for influx of oxygenfree radical producing cells, demonstrated histochemically with3,3′-diaminobenzidine (DAB) at the light microscopical level (ref. 27).A significant influx of oxygen free radical producing cells was observedin dermal sites injected with untreated endotoxin as compared tocontrols (E/S versus C/S p<0.01, Wilcoxon; FIG. 4). This inflammatoryresponse was attenuated at dermal sites injected with endotoxinpretreated with tubular brushborder fragments (E/S versus E/A, p<0.05,Wilcoxon), whereas endotoxin pretreated with tubular fragments pluslevamisole displayed increased pro-inflammatory activity as compared toendotoxin pretreated with tubular fragments alone (E/A versus E/A/L,p<0.025, Wilcoxon). Each test was performed in duplicate on the same ratand results are expressed as the arithmetic means (+/−SD) of 6 rats.These data demonstrate that endotoxin treated with alkaline phosphataseexhibits reduced toxicity in vivo and that alkaline phosphatase may beable to detoxify endotoxin in vivo.

To examine the contribution of endogenous alkaline phosphatase activityin the endotoxin detoxification in vivo, we assessed the effect oflevamisole upon endotoxin-sensitivity in rats, a species relativelyresistant to Gram-negative lipopolysaccharrides. Female PVG rats, 6months of age, received the alkaline phosphatase inhibitor levamisole(Sigma Chemical Co, St.Louis, USA) intraperitoneally (10 mg/kg b.w.), orsaline at t=−24 and t=−1 hour. At t=0, rats received an i.v. challengeof 0.5 mg endotoxin, and blood was collected immediately prior to and att=3, 6, 24 and 48 hours after this injection.

Serum glutamate-pyruvate transaminase activity, reflecting liver damage(an important pathogenic factor in endotoxin-induced death) was assessedin these samples according to the method of Wroblewski and LaDue.Results showed no change in serum transaminase activity after treatmentwith levamisole alone as compared to saline treated rats whereas anincrease was found after the endotoxin challenge (FIG. 5). However, incontrast to the minor increase observed in rats receiving onlyendotoxin, rats pre-treated with levamisole displayed a very strongincrement in serum transaminase activity (p<0.001), demonstrating theinvolvement of endogenous alkaline phosphatase activity in theendotoxin-detoxifying activity of rats in vivo.

The detoxifying activity of alkaline phosphatase was also studied in anexperimental model of septic shock in rats and mice. Thus, rats receivedan intraperitoneal injection of 1.0×10¹⁰ colony forming units (CFU) of awell characterized strain of Escherichia coli bacteria (ATCC 25922)whereas mice received 0.2×10¹⁰ CFU. Inoculation of this dose leads tothe full blown septic shock syndrome, characterized by thrombocytopenia.leucopenia, impaired liver function and reduced (rectal) bodytemperature, within approximately 6 hours.

Most rats, being relatively resistant to endotoxin, survived theinjection of E. coli bacteria (FIG. 7A). However, in combination withthe alkaline phosphatase-inhibitor levamisole (50 mg/kg b.w.administered subcutaneously 2 hours prior to the bacteria) inoculationof Gram-negative bacteria was lethal. Nine out of ten rats died withclinical symptoms of shock. Serum levels of alkaline phosphataseactivity in rats treated with Levamisole were reduced by 50% six hoursafter the injection. Levamisole did not influence survival of rats whichreceived a sublethal dose of Gram-positive bacteria; rats receiving 50mg/kg b.w. Levamisole 2 hours before administration of 1.0×10¹⁰ CFUStaphylococcus aureus nearly all survived (8 rats per group, FIG. 7B).This demonstrates that endogenous alkaline phosphatase activity in ratsis involved in the resistance towards endotoxin of Gram-negativebacteria.

In contrast to rats, nine out of ten mice died from of dose of 0.2×10¹⁰CFU E. coli (FIG. 8). However, animals receiving a singleintraperitoneal injection of 0.15 U purified human placental alkalinephosphatase 2 hours before the administration of bacteria all survivedthe inoculation of this lethal dose (n=10), alkaline phosphatase wasextracted from human placenta with butanol (50% v/v) and purified usinga diethylaminoethyl cellulose column and an affinity column (aCNBr-sepharose^(4B) column with rabbit-anti-human-placental alkalinephosphatase antibodies coupled to it). Thus, alkaline phosphataseappears to be able to detoxify endotoxin in vivo and seems applicablefor the treatment of endotoxic shock.

The circulating half-life of placental alkaline phosphatase isapproximately 7 days in human blood (ref. 31). In rats human placentalalkaline phosphatase is detectable in blood for approximately three days(personal observations), in contrast to intestinal alkaline phosphatasewhich has a circulating half-life of 71/2 minutes (ref. 28). Based onthese data and based upon the results of studies in mice (see above), itcan be concluded that placental alkaline phosphatase is particularlysuitable to serve as an active component in a pharmaceuticalcomposition, in particular a systemically applicable composition such asfor the prevention of sepsis.

The anti-endotoxin activity being based on the dephosphorylatingactivity exhibited by alkaline phosphatase can naturally not be excludedfor other phosphatases, in particular phosphatases with optimum pH invitro at alkaline pH or derivatives of phosphatases having phosphataseactivity. The subject invention is therefore directed at the use of aphosphatase as such or a derivative thereof having phosphatase activityas active component for the preparation of a pharmaceutical compositionfor prophylaxis or therapy of pathology mediated by endotoxin or by aderivative of endotoxin having endotoxic activity. A pharmaceuticalcomposition comprising at least a phosphatase as such or a derivativethereof having phosphatase activity suitable for systemic application orcomprising a vehicle capable of producing phosphatase as such or aderivative of phosphatase having phosphatase activity suitable forsystemic application as active component, said phosphatase or saidderivative having detoxifying activity for an endotoxin or for aderivative of endotoxin having endotoxic activity and further comprisinga pharmaceutically acceptable carrier, falls within the scope of theinvention. In particular a pharmaceutical composition comprisingalkaline phosphatase as such or a derivative thereof having phosphataseactivity or a vehicle capable of producing alkaline phosphatase as suchforms a preferred embodiment of the invention. For use in apharmaceutical composition the phosphatase must be obtainable in asubstantially pure form. In general recombinant DNA techniques canprovide a phosphatase suitable for use in a pharmaceutical compositionaccording to the invention.

To date, four isozymes encoded by four distinct genes have beendescribed. These include the intestinal form, the liver/bone/kidney-type(also present in neutrophils), the placenta-type, and the placental-likeisozyme (present in germinal cells). Both the intestinal form and theliver/bone/kidney-type alkaline phosphatase exhibit endotoxindetoxifying activity, whereas there are no reasons to believe that theother isozymes of alkaline phosphatase are not able to degradeendotoxin. Therefore the subject invention comprises a pharmaceuticalcomposition comprising any such isozyme. In particular an alkalinephosphatase of the placental type is suitable due to the long half lifethereof in vivo.

The following case history illustrates the deleterious consequences of areduced alkaline phosphatase activity in a mammal such as a human. A oneyear old female child suffered from recurrent endotoxaemia. Theseperiods were accompanied with life-threatening symptoms of shock. Upontreatment, recovery was achieved, however, often followed by a relapsewithin a few weeks when endotoxaemia occurred again. In a period of 10months, 12 relapses happened and finally the child died at the age ofone year. The cause of death was diagnosed as septic shock induced byGram-negative bacteria. The cause of the recurrent endotoxaemia itselfwas unknown. Our recent studies showed that in liver and spleen,alkaline phosphatase appeared normal, but enzyme activity was nearlyabsent in the ileum, an organ which normally expresses the highestenzyme activity of the human body. In the light of our finding, areduced alkaline phosphatase activity in such a crucial organ, mayexplain the cause of death. It is easily conceivable that the lack of anendogenous detoxifying mechanism in the intestine results in recurrentendotoxaemia, considering the high content of E. coli in the intestinallumen. Such a deficiency might be adequately treated according to thesubject invention.

Retrospective studies in patients suffering from recurrent endotoxemia(without underlying complications such as malignancies orliver-diseases) showed that serum alkaline phosphatase or a derivativethereof activity correlates with the clinical condition. Thus, whensepsis occurred, invariably a strong decrease, in serum alkalinephosphatase or a derivative thereof activity could be measured, aphenomenon also found in rats, followed by an increase in alkalinephosphatase or a derivative thereof activity in serum within a few days.In a particular case, the inflammatory tissue was removed (partialileum-resection) during a period of serious illness. This caused animmediate rise in serum alkaline phosphatase or a derivative thereofactivity to normal levels. These observations point to an increasedturn-over of the enzyme during Gram-negative bacterial infections andsupport the notion that alkaline phosphatase or a derivative thereofadministration to patients with sepsis may be helpful.

A method for therapy or prophylaxis of pathology mediated by endotoxinor by a derivative of endotoxin having endotoxic activity comprisingadministering to a subject a therapeutic amount of a phosphatase orderivative of phosphatase having phosphatase activity therefore alsofalls within the scope of the invention. Derivatives with optimalactivity around in vivo pH will be preferred. Use of phosphatasederivatives in a method for preparation of a pharmaceutical compositionfor prophylaxis of therapy of pathology mediated by endotoxin or aderivative of endotoxin naturally therefore also falls within the scopeof the invention. An especial preference is expressed for systemicallyapplicable derivatives as such derivatives can be applied in thebloodstream of the subject to be treated as already elucidated forphosphatase as such. For effectivity against Gram-negative bacterialinfections the active component must preferably arrive at any locationwhere a Gram-negative bacterium or it's endotoxin can be located.

Another aspect of the invention lies in the insight that in accordancewith the Brønsted-Lowry classification of acids and bases, polyanionsmay act as weak bases since they are able to bind H⁺. Thus extrapolatingthis to the fact that optimal functioning of proteins or polypeptides isoften pH dependent and that in particular in vitro it has beenillustrated that the incubation medium has to be alkaline for optimalactivity of said proteins or polypeptides, in particular for alkalinephosphatase, we have concluded that in vivo polyanionic sites, forinstance negatively charged sialoglycoproteins associated with cellmembranes may meet the pH demands of such an enzyme or polypeptide.

Alkaline phosphatase is predominantly found in association withplasma-membranes. For example, neutrophils present the enzyme againstthe background of their negatively charged cell membrane instead ofreleasing it into the inflammatory micro-environment. For this reason itis felt that poly-anionic substrates could further contribute tofavourable an ionic conditions in vivo for phosphatase activity ofphosphatases and derivatives thereof normally having an optimum at analkaline pH, in particular for phosphatase activity of alkalinephosphatase.

If negatively charged sugar moieties of endotoxin influence the pHoptimum of this enzyme activity, polycations could be expected tointerfere with this reaction. Therefore, we treated the substrates withthe cations poly-ethyleneimine (PEI) or poly-L-lysin (Lys). Substrateswere preincubated for 30 minutes with either 0.5% PEI, 0.75%poly-L-lysin or distilled water (C). Subsequently, incubations werecarried out as described above and phosphate release was assessed. Bothcations strongly affected dephosphorylation of endotoxin by neutralisingthe negative charges, whereas neither one of them significantlyinfluenced pNPP degradation (FIG. 3). The profound effect of PEI uponendotoxin degradation may be caused by neutralisation of negativeCharges whereas steric inhibition may also add to this effect.Interestingly, poly-L-lysin caused a shift of the pH optimum to a higherlevel, supporting the idea that negatively charged residues in the microenvironment determine the pH optimum.

Additional support for the notion that negatively charged molecules inthe micro-environment influence the pH optimum of alkaline phosphatasewas also derived from experiments with intestinal enzymes.Histo-chemical assessment of the pH optimum of alkaline phosphatase incryostat sections (4 μm) of rat intestine applying the method of Gomoriwith the substrate pNPP, revealed no significant change in stainingintensity when the pH level of the incubation medium was varied from 7.8to 9.8. However, alkaline phosphatase activity in serum, which is shownto be of intestinal origin, exhibits a pH optimum of 9.8 or higher. Animportant difference between intestinal alkaline phosphatase in situ andin serum is the sialoglycoprotein content thereof. Although theintestinal enzymes are embedded in sialated plasma membranes. serumalkaline phosphatase is not surrounded by these polyanions.

The subject invention is therefore also directed at a derivative of aphosphatase having phosphatase activity and comprising a net highercontent of negatively charged moieties than the corresponding nativephosphatase. In particular the invention is directed at such aderivative being derived from a phosphatase with an optimum at alkalinepH such as alkaline phosphatase.

A derivative of a phosphatase according to the invention comprising ahigher content of negatively charged moieties can comprise a highercontent of derivatized alkaline amino moieties than the correspondingnative phosphatase. This can for example be achieved by said derivativecomprising a higher content of negatively charged N-acetylneuraminicacid groups (=sialic acid groups) than the corresponding nativephosphatase. Another possibility lies in the derivative comprising ahigher content of negatively charged acid and/or a reduced number ofbasic moieties than the corresponding native phosphatase as such or incombination with the aforementioned embodiment. In yet anotherembodiment of the invention the derivative of phosphatase can comprise aphosphatase moiety connected to a negatively charged protein orpolypeptide. A suitable example of such a negatively charged protein isa modified negatively charged albumin, e.g. a succinylated albumin. Itis repeated that the disclosed derivatives of phosphatase of WO93/00935, U.S. Pat. Nos. 4,394,370, 4,409,332 and EP-A-0441252 are notderivatives with an increased netto negative charge. The only morenegatively charged derivative disclosed in said literature is therecombinant E. coli alkaline phosphatase with substitution of Ala 103 byAsp. In addition, none of the disclosed derivatives are systemicallyapplicable. A person skilled in the art will know what type ofderivatives are implied by the term systemically applicable. What is ofparticular interest are derivatives that can be used in oral dosageforms, or intravenous solutions or any medicinal dosage form such thatthe active components can enter the bloodstream. Toxic derivatives suchas collagen derivatives are not acceptable.

In a further aspect of the invention a derivative of phosphatase cancomprise at least one modification for increasing the half life of saidderivative in vivo, e.g. by preventing binding to galactose receptors,said modification e.g. being located at the terminal galactose residueof a phosphatase such as alkaline phosphatase. It was already known thatremoval of e.g.serum alkaline phosphatase is mediated by hepaticgalactose receptors (ref. 28) but no attempt to modify a substrate ofsuch a receptor has been taught or suggested previously. Themodification can e.g. be the result of an oxidation or reduction.

The invention not only covers the derivatives as such in the variousembodiments described above but also comprises combinations of thevarious aspects of such embodiments.

With regard to the various embodiments possible for a derivative ofphosphatase WO 92/15316 gives examples of how to modify proteins andpolypeptides in order to provide modified substances with an additionalnet negative charge by derivatisation of their amino groups and/or otherbasic functional groups with a reagent that prevents protonisation ofbasic amino groups and/or other basic functional groups or replaces saidbasic groups by one or more functional groups having a negative charge.The groups to be derived can be histidine and/or lysine residues. Thereagent can be chosen from aldehydes, anhydrides, acid chlorides andisothiocyanates. For serum albumin a suitable reagent is cis-aconitateanhydride. Another suitable protein to be modified and linked tophosphatase to form a derivative according to the invention is α-acidglyco-protein.

It is also possible to create a derivative of alkaline phosphatasehaving optimal phosphatase activity at physiological pH, whichderivative is therefore suitable for use in vivo and such a derivativealso falls within the scope of the invention.

The subject invention not only covers the derivatives as described abovein the various embodiments but also covers a pharmaceutical compositioncomprising at least one such derivative of phosphatase havingphosphatase activity or a vehicle capable of producing such a derivativeof phosphatase having phosphatase activity as active component andfurther comprising a pharmaceutically acceptable carrier.

In particular a pharmaceutical composition, wherein the active componentas described i.e. the phosphatase as such, the derivative of phosphatasewhich is systemically applicable and/or comprises a net negative chargelarger than that of the corresponding native phosphatase or a vehiclecapable of producing the phosphatase or the derivative thereof havingphosphatase activity is embedded in the lipid bilayer of a liposome,preferably in combination with negatively charged membrane constituentsis a preferred embodiment of the invention. In such a composition thephosphatase is a phosphatase having phosphatase activity havingdetoxifying activity for an endotoxin or a derivative thereof.

Use of a phosphatase derivative in any of the embodiments describedabove as active component for the preparation of a pharmaceuticalcomposition for prophylaxis or therapy of pathology mediated byendotoxin or by a derivative of endotoxin having endotoxic activity isalso therefore covered by the subject invention. In particular apharmaceutical composition comprising a negatively charged and/orsystemically applicable derivative of alkaline phosphatase or a vehiclecapable of delivering and/or inducing synthesis of the alkalinephosphatase of the derivative in any of the described embodiments formsa preferred embodiment of the invention. A systemically acceptablepharmaceutical composition is preferred.

A method for therapy or prophylaxis of pathology mediated by endotoxinor by a derivative thereof having endotoxic activity comprisingadministering to a subject a therapeutic amount of such a pharmaceuticalcomposition or any derivative of a phosphatase having phosphataseactivity and a pharmaceutically acceptable carrier also falls within thescope of the invention. In particular the subject invention is alsodirected at a method for preventing occurrence of a pathology mediatedby endotoxin or a derivative thereof having endotoxic activity followingtransplant or transfusion, said method comprising subjecting thematerial to be transplanted or transfused to treatment before and/orduring and/or after transplant or transfusion with one of the followingcomponents:

phosphatase as such having detoxifying activity for an endotoxin or fora derivative of endotoxin having endotoxic activity, said phosphatasepreferably being alkaline phosphatase, more preferably human alkalinephosphatase;

a derivative of a phosphatase having phosphatase activity according toat least one of the embodiments of the invention as described above;

a vehicle capable of delivering and/or inducing synthesis or phosphataseactivity of the phosphatase or the phosphatase derivative havingdetoxifying activity for an endotoxin or for a derivative of endotoxinhaving endotoxic activity, said phosphatase preferably being alkalinephosphatase;

a vehicle capable of delivering and/or inducing synthesis or phosphataseactivity of a derivative of phosphatase according to the invention asdescribed above, as such or as active component of a pharmaceuticalcomposition. Preferably the pharmaceutical composition has a formrendering the active component capable of entering the blood stream.

To test whether the endotoxin detoxifying mechanism is upregulated inthe presence of this bacterial product, human neutrophils were isolated,pre-incubated with endotoxin and assayed for alkaline phosphataseactivity. Thus, neutrophils were isolated from normal human volunteersaccording to standard methods and collected in sterile medium.Neutrophils were not activated during the isolation procedure asassessed by measurements of superoxide anion production by these cells.Subsequently, cells (0.9×10⁷ cells/ml) were incubated at 37° C. inbuffer supplemented with endotoxin (20 pg/ml) or saline. After 30minutes, alkaline phosphatase activity was assayed in these samplesaccording to standard methods at pH 9.8 with pNPP as substrate.Phosphatase activity was measured with and without levamisole (1 mM)added to the medium. Results show an increase of 335% in neutrophilicalkaline phosphatase activity induced by endotoxin (FIG. 6), which is inaccordance with the proposed function of this enzyme.

The invention also covers a pharmaceutical composition comprising atleast one of the following components:

a substance for stimulating phosphatase activity, of a phosphatase or aderivative thereof having detoxifying activity for an endotoxin or for aderivative of endotoxin having endotoxic activity, preferably forstimulating alkaline phosphatase activity;

a vehicle capable of delivering and/or inducing synthesis of a substancefor stimulating phosphatase activity of a phosphatase or for aderivative thereof having detoxifying activity for an endotoxin or aderivative of an endotoxin having endotoxic activity, preferably forstimulating alkaline phosphatase activity as active component andfurther comprising a pharmaceutically acceptable carrier, saidpharmaceutical composition being systemically applicable.

The invention also covers a pharmaceutical composition comprising atleast one of the following components:

a substance for stimulating phosphatase activity, of a phosphatase or aderivative thereof having detoxifying activity for an endotoxin or for aderivative of endotoxin having endotoxic activity, preferably forstimulating alkaline phosphatase activity;

a vehicle capable of delivering and/or inducing synthesis of a substancefor stimulating phosphatase activity of a phosphatase or for aderivative thereof having detoxifying activity for an endotoxin or aderivative of an endotoxin having endotoxic activity, preferably forstimulating alkaline phosphatase activity as active component andfurther comprising a pharmaceutically acceptable carrier in combinationwith a further active component being a phosphatase as such or aderivative of a phosphatase having phosphatase activity or a vehiclecapable of delivering and/or inducing synthesis of said phosphataseand/or said derivative, with the proviso that the pharmaceuticalcomposition does not comprise (alkaline) phosphatase as derivative withcollagen and/or demineralized bone, i.e. does not comprise apharmaceutical composition as described by Beersten et al. forosteogenisis in situ of the desired bone formation. A pharmaceuticalcomposition comprising a derivative of phosphatase having a net negativecharge in comparison to the corresponding native phosphatase or avehicle capable of delivering and/or inducing synthesis of saidderivative as one of the active components in combination with thesubstance or substance producing vehicle, said substance being capableof stimulating phosphatase activity is included within the scope ofinvention. The activating effect of the natural detoxifying action ofphosphatase, preferably endogenous phosphatase, in particular ofalkaline phosphatase can thus be stimulated further and provide a meansof defense against the negative pathological symptoms caused byendotoxins or derivatives thereof having endotoxic activity. Inparticular the substance for stimulating phosphatase activity,preferably for stimulating alkaline phosphatase activity can be selectedfrom one or more of the following: an endotoxin, a substance havingendotoxic activity, granulocyte colony stimulating factor (G-CSF),retinoic acid, a glucocorticoid and any other cytokines or substancesknown to stimulate phosphatase activity (ref. 13, 29 and 30).

The subject invention also covers the use of any of the aforementionedactive components for stimulating phosphatase activity as activecomponent for preparation of a pharmaceutical composition forprophylaxis or therapy of pathology mediated by endotoxin or aderivative of endotoxin having endotoxic activity. It also covers theuse of said active component for stimulating phosphatase activity incombination with phosphatase as such or any derivative of phosphatasehaving phosphatase activity or any vehicle capable of delivering and/orinducing the synthesis of said derivative for preparation of such apharmaceutical composition.

A method for preventing occurrence of a pathology mediated by endotoxinor a derivative thereof having endotoxic activity, said methodcomprising stimulating phosphatase activity in particular by increasingendogenous alkaline phosphatase activity, for example by increasingproduction thereof in a subject by administering at least one of theaforementioned components as such or as active component of apharmaceutical composition to the subject also falls within the scope ofthe invention. As does a method for preventing occurrence of a pathologymediated by endotoxin or a derivative thereof having endotoxic activityfollowing transplant or transfusion, said method comprising subjectingthe material to be transplanted or transfused to treatment stimulatingphosphatase activity, in particular by increasing the activity ofendogenous alkaline phosphatase, for example by increasing productionthereof of said material by administering at least one of the followingcomponents:

a substance for stimulating phosphatase activity, in particular byincreasing the activity of endogenous phosphatase or a derivativethereof having detoxifying activity for an endotoxin or for a derivativeof an endotoxin having endotoxic activity;

a vehicle capable of delivering and/or inducing synthesis of thesubstance for stimulating phosphatase activity as such or as activecomponent of a pharmaceutical composition to the subject. A suitableembodiment of this aspect of the invention can be found in a method forincreasing the presence of alkaline phosphatase, preferably endogenousalkaline phosphatase in the body, the tissue or the body fluid of ananimal or human comprising application of at least one of theaforementioned components for stimulating phosphatase activity as suchor as active component of a composition.

In summary from the above described experiments it can be concluded thatenzyme preparations based upon alkaline phosphatase activity are able todephosphorylate endotoxin thereby attenuating the toxicity of thismolecule in vitro and in vivo. The physiological activity of this enzymeis most prominent when associated with negatively charged residues whichprovide the proper micro environmental conditions. Furthermore, alkalinephosphatase activity can be upregulated in cells of the host-defencesystem, providing a natural barrier against endotoxin.

The subject invention also covers a pharmaceutical compositioncomprising at least one of the following components:

phosphatase as such having phosphatase activity in vivo in or on bone,said phosphatase preferably being alkaline phosphatase, more preferablyhuman (alkaline) phosphatase and preferably being recombinantphosphatase;

a systemically acceptable derivative of the alkaline phosphatase, saidderivative having said phosphatase activity in vivo in or on bone and/ora derivative;

a vehicle capable of delivering and/or inducing synthesis or phosphataseactivity of said phosphatase as such an/or said derivative and/or thederivative, as active component in vivo and further comprising apharmaceutically acceptable carrier. In vivo in this context implies ina macro environment having physiological pH, i.e. a pH of 7-8. Asuitable embodiment of such a pharmaceutical composition comprises theactive component embedded in the lipid bilayer of a liposome, preferablyin combination with negatively charged membrane constituents.

The subject invention further comprises use of at least one of thefollowing components:

a phosphatase as such having phosphatase activity in vivo, preferably inor on bone, more preferably alkaline phosphatase and more preferablyhuman alkaline phosphatase and preferably being recombinant phosphatase;

a derivative of alkaline phosphatase having phosphatase activity invivo, preferably in or on bone;

a vehicle capable of delivering and/or inducing synthesis of saidphosphatase as such and/or said derivative as active component in vivofor preparation of a pharmaceutical composition for prophylaxis ortherapy of metabolic bone diseases, such as osteoporosis andosteomalacia.

Use of at least one of the following components:

phosphatase as such having phosphatase activity in vivo, preferably inor on bone more preferably alkaline phosphatase and more preferablyhuman (alkaline) phosphatase and preferably being recombinantphosphatase,

a derivative of phosphatase having phosphatase activity in vivopreferably in or on bone, said derivative being a systemicallyacceptable derivative of alkaline phosphatase having phosphataseactivity in vivo and/or a derivative, preferably with increased netnegative charge in comparison to the corresponding native phosphatase;

a vehicle capable of delivering and/or inducing synthesis or phosphataseactivity of said phosphatase as such and/or said derivative,

as active component for preparation of a pharmaceutical composition forprophylaxis or therapy of pathology requiring increased bone formationsuch as stimulating mending of broken bone and prophylaxis or therapy ofmetabolic bone disorders, such as osteoporosis and osteomalacia alsofalls within the scope of invention.

Also a pharmaceutical composition comprising at least one of thefollowing components:

a substance for stimulating phosphatase activity, preferably of anendogenous phosphatase or a derivative thereof, said phosphatase orderivative having detoxifying activity for an endotoxin and/or for aderivative of endotoxin having endotoxic activity and/or saidphosphatase or derivative having phosphatase activity in vivo, inparticular in or on bone, said substance preferably being a substancefor stimulating alkaline phosphatase activity, more preferably human(alkaline) phosphatase;

a vehicle capable of delivering and/or inducing synthesis of saidsubstance for stimulating phosphatase activity, more preferably human(alkaline) phosphatase activity, as active component and furthercomprising a pharmaceutically acceptable carrier, said pharmaceuticalcomposition being systemically applicable falls within the scope of theinvention. The invention is also directed at a pharmaceuticalcomposition comprising at least one of the following components:

a substance for stimulating phosphatase activity, preferably of anendogenous phosphatase or a derivative thereof, said phosphatase orderivative having detoxifying activity for an endotoxin and/or for aderivative of endotoxin having endotoxic activity and/or saidphosphatase or derivative having phosphatase activity in vivo, inparticular in or on bone, said substance preferably being a substancefor stimulating alkaline phosphatase activity, more preferably human(alkaline) phosphatase;

a vehicle capable of delivering and/or inducing synthesis of saidsubstance for stimulating phosphatase activity, more preferably human(alkaline) phosphatase activity, as active component and furthercomprising a pharmaceutically acceptable carrier, in combination with atleast one of the active components,

a phosphatase as such having phosphatase activity in vivo, preferably inor on bone, more preferably alkaline phosphatase and more preferablyhuman alkaline phosphatase and preferably being recombinant phosphatase;

a derivative of (alkaline) phosphatase having phosphatase activity invivo, preferably in or on bone;

a vehicle capable of delivering and/or inducing synthesis of saidphosphatase as such and/or said derivative as active component in vivoor at least one of the following components;

a phosphatase as such having detoxifying activity for an endotoxinand/or for a derivative thereof having endotoxic activity, saidphosphatase preferably being alkaline phosphatase, more preferably humanalkaline phosphatase and said phosphatase preferably being recombinantphosphatase;

a derivative of the phosphatase having phosphatase activity for anendotoxin and/or for a derivative thereof, having endotoxic activity,said phosphatase preferably being alkaline phosphatase, more preferablyhuman alkaline phosphatase and said phosphatase preferably beingrecombinant phosphatase, said derivative preferably being a systemicallyacceptable derivative of a phosphatase and/or being a derivativepreferably having an increased net negative charge in comparison to thecorresponding native phosphatase;

a vehicle capable of delivering and/or inducing synthesis or phosphataseactivity of said phosphatase as such or the derivative of phosphatase asactive component.

The substance for stimulating alkaline phosphatase activity can besuitably selected from one or more of the following: an endotoxin, asubstance having endotoxic activity, granulocyte colony stimulatingfactor (G-CSF), retinoic acid, a glucocorticoid. Any other suchsubstances known to a person skilled in the art can also be used toobtain the desired stimulation of (alkaline) phosphatase activityaccording to the invention.

In a pharmaceutical composition comprising a substance for stimulatingphosphatase activity or a vehicle capable of delivering such a substanceas described above, the further active components can be any of thecomponents;

a phosphatase as such having detoxifying activity for an endotoxinand/or for derivatives thereof having endotoxic activity, saidphosphatase preferably being alkaline phosphatase, more preferably humanalkaline phosphatase and said phosphatase preferably being recombinantphosphatase;

a derivative of the phosphatase, said derivative having the phosphataseactivity and said derivative being systemically acceptable preferablyhaving an increased negative charge in comparison to the correspondingnative phosphatase;

a vehicle capable or delivering and/or inducing synthesis of phosphataseactivity of said phosphatase as such and/or said derivative;

phosphatase as such having phosphatase activity in vivo in or on bone,said phosphatase preferably being alkaline phosphatase, more preferablyhumen (alkaline) phosphatase and preferably being recombinantphosphatase;

a systemically acceptable derivative of the alkaline phosphatase, saidderivative having said phosphatase activity in vivo in or on bone and/ora derivative;

a vehicle capable of delivering and/or inducing synthesis or phosphataseactivity of said phosphatase as such and/or said derivative.

In any of the embodiments disclosed the pharmaceutical composition cancomprise the active component embedded in the liquid bilayer of aliposome, preferably in combination with negatively charged membrainconstituents. Preferably a pharmaceutical composition will besystemically acceptable.

The subject invention is also directed at use of at least one of theactive components of a pharmaceutical composition comprising a substancefor stimulating phosphatase activity or a vehicle for delivering and/orinducing synthesis of said substance as active component for preparationof a pharmaceutical composition for prophylaxis or therapy of pathologymediated by an endotoxin and/or by a derivative of endotoxin havingendotoxic activity. Also the use of at least one of the just mentionedactive components as active component for preparation of apharmaceutical composition for prophylaxis or therapy of metabolic bonediseases, such as osteoporosis and osteomalacia falls within the scopeof the invention. In addition, use of at least one of the activecomponents of a pharmaceutical composition as described herein incombination with at least one of the active components of apharmaceutical composition having a substance for stimulatingphosphatase activity, a vehicle capable of delivering and/or inducingsynthesis of a substance for stimulating phosphatase activity and apharmaceutically acceptable carrier as active component for preparationof a pharmaceutical composition for prophylaxis or therapy of pathologyrequiring increased bone formation such as stimulating mending of brokenbones and prophylaxis or therapy of metabolic bone diseases such asosteoporosis and osteomalacia falls within the scope of the invention.Also the invention is directed at a method for prophylaxis or therapy ofmetabolic bone disease such as osteoporosis and osteomalacia requiringincreased bone formation, said method comprising application of at leastone of a phosphatase, a derivative of an alkaline phosphatase havingphosphatase activity in vivo and a vehicle to the subject to be treated.A method for prophylaxis or therapy of pathology requiring increasedbone formation such as stimulating mending of broken bone andprophylaxis or therapy of metabolic bone disease such as osteoporosisand osteomalacia, said method comprising administering at least one ofthe active components of a pharmaceutical composition as describedherein as such or as active component of a composition to the subject tobe treated as well as a method of treatment of pathology associated withrapid bone formation such as osteosarcoma, said method comprisingdecreasing or inhibiting alkaline phosphatase activity, preferably in atarget specific manner, i.e. at the location where said pathology occursforms part of the invention. In all the methods disclosed the activecomponents can be systemically applicable. In addition to the previouslymentioned pharmaceutical compositions a pharmaceutical compositioncomprising at least one substance capable of decreasing or inhibitingphosphatase activity and/or the concentration of phosphatase inparticular alkaline phosphatase (activity), said substance preferablybeing targeted to act at a location where undesired bone formation is tobe prevented is covered by the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Alkaline phosphatase activity in cryostat sections of ratintestine (A) and kidney (B) as demonstrated at pH 9.0 with thesubstrate β-glycerophosphate. FIG. C and D show phosphatase activity insections of intestine (C) and kidney (D) as demonstrated with endotoxinas substrate at pH 7.4. Significant dephosphorylation of endotoxin isfound along intestinal crypts (FIG. C) and in tubular brushborders ofthe kidney (FIG. D), corresponding with the localization of alkalinephosphatase activity (FIG. A & B). Furthermore, in intestinal sectionsthis activity is reduced by addition of L-phenyl-alanine (FIG. E),whereas endotoxin dephosphorylation in kidney sections is completelyinhibited by levamisole (FIG. F). Abbreviation: m=medulla.Magnification: 35× (A,B), 140× (C,E) and 56× (D,F).

FIG. 2 Phosphatase activity, expressed as inorganic phosphate (Pi)release per hour, in suspensions of tubular brushborders at different pHlevels with endotoxin as substrate. Phosphatase activity with thesubstrate para-nitrophenolphosphate (pNPP) is shown in the upper leftcorner. Tubular brushborder fragments were isolated from the cortex ofPVG rat kidneys and added to 250 μl 2-amino-2-methyl-1,3-propanediolbuffer at different pH levels, containing E. coli endotoxin (1.25 mg/ml)or pNPP (0.5 mg/ml). 2 mM MgCl₂ was added immediately before incubation.Dashed lines indicate phosphatase activity in the presence of levamisole(0.2 mM). After one hour incubation at 37° C., inorganic phosphateconcentrations were assessed as described previously (ref. 29). Resultsare expressed as arithmetic means (±SD) of 6 assays, each assay wasperformed in duplicate. Results show that maximal dephosphorylation ofendotoxin occurs at pH 8.8, whereas dephosphorylation of pNPP shows asteady increase to pH 9.8.

FIG. 3 Phosphatase activity, expressed as phosphate (Pi) release perhour, in suspensions of tubular brushborders at different pH levels withendotoxin (FIG. A) or PNPP (FIG. B) as the substrate. Substrates werepreincubated for 30 minutes with either 0.5% poly-ethyleneimine (PEI),0.75% poly-L-Lysin (Lys) or distilled water (C). Subsequently,incubations were carried out as described at FIG. 2. Results areexpressed as the arithmetic means of 4 assays, each assay was performedin duplicate (±SD).

FIG. 4 To assess endotoxin toxicity in vivo, a localizedShwartzman-reaction (ref. 16) was elicited in the skin of PVG rats atdifferent locations on the back. Prior to injection, all media wereincubated (1 hour, 37° C.) with 6 μg tubular brushborder fragmentscontaining alkaline phosphatase activity (A), or with 0.9% saline (S).When indicated, the alkaline phosphatase inhibitor levamisole (L) wasadded (final concentration 1.0 mM). Two hours after the intradermalinjections, dermal sites were analyzed for influx of oxygen free radicalproducing cells, demonstrated histochemically with 3,3′-diaminobenzidine(DAB) at the light-microscopical level¹⁷. Each test was performed induplicate on the same rat and results are expressed as the arithmeticmean (±SD) of 6 rats.

FIG. 5 Effect of eridotoxin upon serum levels of glutamate-pyruvatetransaminase (GPT) activity, which reflects damage of liver cells, inPVG rats. Part of the animals were pre-treated with levamisole (L; 10mg/kg b.w.) at t=−24 and −1 hr. At t=0 rats received either 0.5 mgendotoxin from E. coli (E) intravenously or 0.5 ml saline. Results areexpressed as arithmetic means (±SD of 4 rats) per group.

FIG. 6 Effect of endotoxin upon alkaline phosphatase (AP) activity ofhuman neutrophils. Neutrophils were isolated from whole blood accordingto standard procedures and incubated in 0.9% saline with or withoutendotoxin from E. coli (20 pg/ml) for 30 minutes at 37° C. Subsequently,phosphatase activity of these cells was measured, using paranitrophenolphosphate as substrate at pH 9.8. Levamisole (1 mM) was added to confirmthe involvement of alkaline phosphatase in phosphate release.

FIG. 7A illustrates the percentage of survival of rats after injectionof E. coli (solid line), Levamisole (Le) (dashed line with triangles) orboth (dashed line with circles) along the X-axis against time in hoursalong the Y-axis.

FIG. 7B illustrates the percentage of survival of rats after injectionof Staphylococcus aureus (solid line), Levamisole (Le) (dashed line withtriangles) or both (dashed line with circles).

FIG. 8 illustrates the survival of mice after injection of E. coli withalkaline phosphatase treatment (dashed line) and without alkalinephosphatase (solid line).

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What is claimed is:
 1. A method of dephosphorylating an endotoxincomprising: administering to a mammal a composition comprising a firstalkaline phosphatase having dephosphorylating activity in vivo in anamount sufficient to dephosphorylate endotoxin produced by bacteria invivo, optionally, a vehicle comprising a second alkaline phosphatasewhich may be identical to said first alkaline phosphatase, havingdephosphorylating activity in vivo, wherein said second alkalinephosphatase is obtained from an isolated, purified recombinant DNAsequence encoding an alkaline phosphatase isozyme havingdephosphorylating activity in vivo and; a pharmaceutically acceptablecarrier, thereby dephosphorylating the endotoxin.
 2. The methodaccording to claim 1, wherein at least one member of the groupconsisting of said first alkaline phosphatase and said second alkalinephosphatase comprises a human alkaline phosphatase.
 3. The methodaccording to claim 2, wherein said human alkaline phosphatase compriseshuman placental alkaline phosphatase.
 4. The method according to claim1, wherein said vehicle comprises liposomes.
 5. The method according toclaim 1, wherein said composition is administered by injection.